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61.
Kusumoto R Dawut L Marchetti C Wan Lee J Vindigni A Ramsden D Bohr VA 《Biochemistry》2008,47(28):7548-7556
Werner syndrome is a rare human disease characterized by the premature onset of aging-associated pathologies, cancer predisposition, and genomic instability. The Werner protein (WRN), which is defective in Werner syndrome ( WS) patients, belongs to the RecQ family helicases and interacts with several DNA metabolic proteins, including DNA repair factors and telomere associated proteins. Nonhomologous end-joining (NHEJ) is an important pathway in the repair of DNA double strand breaks (DSBs), and the DNA-PK complex, composed of the heterodimer Ku 70/86 and the DNA-PK catalytic subunit (DNA-PKcs), together with the XRCC4-DNA ligase IV complex (X4L4), are major factors. One of the most prominent protein interactions of WRN is with Ku 70/86, and it is possible that WRN is involved in NHEJ via its associations with Ku 70/86 and DNA-PKcs. This study demonstrates that WRN physically interacts with the major NHEJ factor, X4L4, which stimulates WRN exonuclease but not its helicase activity. The human RecQ helicase, BLM, which possesses only helicase activity, does not bind to X4L4, and its helicase activity is not affected by X4L4. In a DNA end-joining assay, we find that a substrate, which is processed by WRN, is ligated by X4L4, thus further supporting the significance of their functional interaction. 相似文献
62.
Julia Nonnenmann Renate Stirner Julia Roider Maria C. Jung Kathrin Schr?dl Johannes R. Bogner Rika Draenert 《Journal of virology》2014,88(13):7678-7682
Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunosuppressive function. Compared to the level in healthy controls (HC), no elevation of MDSC in chronic hepatitis C (cHEP-C) patients was found, and there was no difference in MDSC based on genotype or viral load (P > 0.25). Moreover, MDSC of cHEP-C patients inhibited CD8 T cell function as efficiently as MDSC of HC did. Since we detected neither quantitative nor qualitative differences in MDSC of cHEP-C patients relative to those of HC, we postulate that MDSC in peripheral blood are most likely not significant regarding immune dysfunction in cHEP-C. 相似文献
63.
Mating disruption method against the vine mealybug,Planococcus ficus: effect of sequential treatment on infested vines
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Rakefet Sharon Tirtza Zahavi Tamar Sokolsky Carmit Sofer‐Arad Maor Tomer Rika Kedoshim Ally R. Harari 《Entomologia Experimentalis et Applicata》2016,161(1):65-69
The vine mealybug, Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), is a major pest of vineyards. Here, we tested the efficacy of the mating disruption method against the pest when applied during one or two successive years in high and low infestation levels. Following 1 year of treatment, at low initial infestation levels a shutdown of pheromone traps was observed, along with a significant reduction in infested vines. With initially high infestation levels, a gradual reduction in infested vines was observed, with a trap shutdown seen only after the second year of pheromone application. We discuss the implications of the male mating disruption method for this pest in which the wingless females are aggregated with limited movement among vines, offering multiple mating opportunities for the flying male. 相似文献
64.
Shizuo Narimatsu Rika Kato Toshiharu Horie Satoshi Ono Michio Tsutsui Yoshiyasu Yabusaki Shigeru Ohmori Mitsukazu Kitada Takao Ichioka Noriaki Shimada Ryuichi Kato Tsutomu Ishikawa 《Chirality》1999,11(1):1-9
The enantioselectivity of 4‐hydroxylation of bunitrolol (BTL), a β‐adrenoceptor blocking drug, was studied in microsomes from human liver, human hepatoma (Hep G2) cells expressing CYP2D6, and lymphoblastoid cells expressing CYP2D6. Kinetics in human liver microsomes showed that the Vmax value for (+)‐BTL was 2.1‐fold that of (−)‐BTL, and that the Km value for (+)‐BTL was lower than that for the (−)‐antipode, resulting in the intrinsic clearance (Vmax/Km) of (+)‐BTL being 2.1‐fold over its (−)‐antipode. CYP2D6 (CYP2D6‐met) expressed in Hep G2 cells had a methionine residue at position 373 of the amino acid sequence and a rat‐type N‐terminal peptide (MELLNGTGLWSM) instead of the human‐type (MGLEALVPLAVIV), and showed enantioselectivity of [(+)‐BTL < (−)‐BTL] for the rate of BTL 4‐hydroxylation. In contrast, enantioselectivity [(+)‐BTL > (−)‐BTL] for Hep G2‐CYP2D6 (CYP2D6‐val) with a human‐type N‐terminal peptide that had a valine residue at 374, which corresponds to the methionine of the CYP2D6‐met variant, was the same as that for human liver microsomes. We further confirmed that CYP2D6‐met and CYP2D6‐val expressed in human lymphoblastoid cells, both of which have methionine and valine, respectively, at position 374 and a human‐type N‐terminal peptide, exhibited the same enantioselectivities as those obtained from CYP2D6‐met and CYP2D6‐val expressed in the Hep G2 cell system. These results indicate that the amino acid at 374 of CYP2D6 is one of the key factors influencing the enantioselectivity of BTL 4‐hydroxylation. Chirality 11:1–9, 1999. © 1999 Wiley‐Liss, Inc. 相似文献
65.
Functional analysis of protein disulfide isomerases in blood feeding, viability and oocyte development in Haemaphysalis longicornis ticks 总被引:1,自引:0,他引:1
Liao M Boldbaatar D Gong H Huang P Umemiya R Harnnoi T Zhou J Tanaka T Suzuki H Xuan X Fujisaki K 《Insect biochemistry and molecular biology》2008,38(3):285-295
Three protein disulfide isomerases from Haemaphysalis longicornis ticks (designated as HlPDI-1, HlPDI-2, and HlPDI-3) were previously identified. In order to further analyze their biological functions, the dsRNA of each HlPDI gene and one dsRNA combination of HlPDI-1/HlPDI-3 were separately injected into female ticks. Reduction of gene and protein expression of HlPDIs by RNA interference (RNAi) was demonstrated by real-time PCR, RT-PCR and Western blot analysis. In single dsRNA-injected groups, HlPDI-1 RNAi impacted tick blood feeding and oviposition, HlPDI-2 RNAi impacted tick viability and HlPDI-3 RNAi had no significant impact by itself. However, the injection of a combination of HlPDI-1/HlPDI-3 dsRNA had synergistic effects on tick viability. Furthermore, the midgut and cuticle were severely damaged in HlPDI-2 dsRNA-injected ticks and HlPDI-1/HlPDI-3 dsRNA-injected ticks, respectively, and disruption of HlPDI genes led to a significant reduction of disulfide bond-containing vitellogenin (Vg) expression in ticks. These results indicate that PDIs from H. longicornis are involved in blood feeding, viability and oocyte development, probably by mediating the formation of disulfide bond-containing proteins of the ticks and the formation of basement membrane and cuticle components such as extracellular matrix (ECM). This is the first report on the functional analysis of PDI family molecules as well as the interactions of PDI and other molecules in blood-feeding arthropods. 相似文献
66.
Hidenori Ito Kimie Atsuzawa† Rika Morishita Nobuteru Usuda† Kaori Sudo Ikuko Iwamoto Kosuke Mizutani‡ Ritsuko Katoh-Semba Yoshinori Nozawa‡ Tomiko Asano Koh-ichi Nagata 《Journal of neurochemistry》2009,108(4):867-880
Septins, a conserved family of GTP/GDP-binding proteins, are present in organisms as diverse as yeast and mammals. We analyzed the distribution of five septins, Sept6, Sept7, Sept8, Sept9 and Sept11, in various rat tissues by western blot analyses and found all septins to be expressed in brain. We also examined the developmental changes of expression of these septins in the rat brain and found that the level of Sept8 increased during post-natal development. Morphological analyses revealed that Sept8 is enriched at pre-synapses. Using yeast two-hybrid screening, we identified vesicle-associated membrane protein 2 (VAMP2), a soluble N -ethylmaleimide-sensitive factor attachment protein receptor (SNARE), as an interacting protein for Sept8. Synaptophysin is reported to associate with and recruit VAMP2 to synaptic vesicles and dissociate prior to forming the SNARE complex consisting of VAMP2, syntaxin and synaptosome-associated protein of 25 kDa. We showed that Sept8 suppresses the interaction between VAMP2 and synaptophysin through binding to VAMP2. In addition, we found that Sept8 forms a complex with syntaxin1A, and the Sept8-VAMP2 interaction is disrupted by synaptosome-associated protein of 25 kDa. These results suggest that Sept8 may participate in the process of the SNARE complex formation and subsequent neurotransmitter release. 相似文献
67.
Asada M Shinomiya M Suzuki M Honda E Sugimoto R Ikekita M Imamura T 《Biochimica et biophysica acta》2009,1790(1):40-48
BACKGROUND: Many fibroblast growth factor family proteins (FGFs) bind to the heparan sulfate/heparin (HP) subtypes of sulfated glycosaminoglycans (GAGs), and a few have recently been reported to also interact with chondroitin sulfate (CS), another sulfated GAG subtype. METHODS: To gain additional insight into this interaction, we prepared all currently known FGFs (i.e., FGF1-FGF23) and assessed their affinity for HP, CS-B, CS-D and CS-E. In addition, midkine, hepatocyte growth factor and pleiotrophin were studied as other known HP-binding proteins. RESULTS: We found that members of the FGF19 subfamily (i.e., FGF15, 19, 21 and 23) had little or no affinity for HP; all of the other secretable growth factors tested had strong affinities for HP, as was indicated by the finding that their elution from HP-Sepharose columns required 1.0-1.5 M NaCl. We also found that FGF3, 6, 8 and 22 had strong affinities for CS-E, while FGF5 had a moderate affinity for CS-D. The interactions between FGFs and GAGs thus appear to be more diverse than previously understood. GENERAL SIGNIFICANCE: This is noteworthy, as the differential interactions of these growth factors with GAGs may be key determinants of their specific biological activities. 相似文献
68.
Hideki Watanabe Hiroyuki Matsumaru Ayako Ooishi YanWen Feng Takayuki Odahara Kyoko Suto Shinya Honda 《The Journal of biological chemistry》2009,284(18):12373-12383
Protein-protein interaction in response to environmental conditions enables
sophisticated biological and biotechnological processes. Aiming toward the
rational design of a pH-sensitive protein-protein interaction, we engineered
pH-sensitive mutants of streptococcal protein G B1, a binder to the IgG
constant region. We systematically introduced histidine residues into the
binding interface to cause electrostatic repulsion on the basis of a rigid
body model. Exquisite pH sensitivity of this interaction was confirmed by
surface plasmon resonance and affinity chromatography employing a clinically
used human IgG. The pH-sensitive mechanism of the interaction was analyzed and
evaluated from kinetic, thermodynamic, and structural viewpoints.
Histidine-mediated electrostatic repulsion resulted in significant loss of
exothermic heat of the binding that decreased the affinity only at acidic
conditions, thereby improving the pH sensitivity. The reduced binding energy
was partly recovered by “enthalpy-entropy compensation.” Crystal
structures of the designed mutants confirmed the validity of the rigid body
model on which the effective electrostatic repulsion was based. Moreover, our
data suggested that the entropy gain involved exclusion of water molecules
solvated in a space formed by the introduced histidine and adjacent tryptophan
residue. Our findings concerning the mechanism of histidine-introduced
interactions will provide a guideline for the rational design of pH-sensitive
protein-protein recognition.Molecular interactions govern a number of biological processes, including
metabolism, signal transduction, and immunoreaction. A better understanding of
the molecular basis for these interactions is crucial for a complete
elucidation of biological phenomena and redesign of interactions for drug
discovery and industrial biotechnology applications. Interactions between
biomolecules are generally characterized by their affinity, specificity, and
environmental responsiveness, such as sensitivity to pH. Such pH-dependent
ligand binding enables biological processes to function in an “on and
off” manner in response to environmental conditions, resulting in
sophisticated systems of regulation (e.g. pheromone production
(1,
2), immune systems
(3-5),
and mechanisms of virus survival
(6)).From an industrial perspective, pH sensitivity is advantageous to various
fields, such as drug delivery systems for medications
(7), biosensing techniques
(8,
9), and affinity chromatography
(10,
11). Although structure-based
protein design is a promising technique for improving molecular function
(12-15),
it is yet difficult to specifically modulate pH sensitivity of a
protein-protein interaction without an associated loss of inherent function
and/or structural stability. Some naturally occurring proteins undergo
substantial conformational change by pH shift, thereby achieving pH-dependent
binding for small molecules (2,
4,
16,
17). However, artificial
design of an equivalent mechanism involving conformational change is highly
problematic. Indeed, proteins have multiple degrees of freedom and consist of
a large number of atoms. Therefore, given that the resulting protein must
maintain both its innate binding ability and structural stability, the system
appears too complicated for rational design. By contrast to the method based
on conformational change, a rigid body-based model (i.e. introduction
of electrostatic repulsion or attraction into a binding interface between
rigid protein domains) could be a more promising approach for pH switching.
Naturally occurring proteins with pH sensitivity generally conserve histidine
residues
(18-21),
which function as a pH switch at slightly acidic conditions (pH ∼6.5) near
the pKa of the histidine side chain. In the presence of a
histidine residue at a binding interface, dissociation under acidic conditions
would be driven by electrostatic repulsion between rigid domains without
conformational change (Fig. 1).
This mechanism is rather simple and applicable to protein engineering
(22,
23). However, to our
knowledge, it still remains unclear how systematic design should be carried
out and, in particular, how histidine-mediated electrostatic repulsion
influences protein-protein interactions. Indeed, very little experimental data
are available for the molecular basis of histidine-introduced protein
binders.Open in a separate windowFIGURE 1.A schematic model for introduction of histidine-mediated electrostatic
repulsion into the binding interface between protein G (GB) and
Fc. Protein G residues positioned closely to basic side chains (depicted
as B) on Fc were systematically identified by distance calculations
and mutated into histidine to cause electrostatic repulsion under acidic
conditions. The inset shows an example of candidate positions for the
mutation.To better understand the design methodology for a pH-sensitive
protein-protein interaction, we generated a number of pH-sensitive
streptococcal protein G B1
(24) mutants by rationally
introducing histidine residues onto the binding surface. Protein G, a
bacterial Fc (fragment of crystallizable region) receptor to the constant
region of IgG, has been used as an affinity chromatography binder for antibody
immobilization and purification. Protein G has an acidic pH optimum for
binding relative to another bacterial Fc receptor, Staphylococcus
aureus protein A. The harsh elution conditions are likely to induce
acidic conformational changes in antibodies
(25,
26) during the purification
procedure, causing aggregation that is problematic for pharmaceutical
applications. The usefulness of the histidine-mediated electrostatic repulsion
for antibody purification was examined by constructing affinity chromatography
columns. Using the designed mutants, we analyzed the molecular basis of the
histidine-mediated interaction from a kinetic, thermodynamic, and structural
perspective. The observed data revealed functional and structural consequences
for the introduction of histidine residues. Analysis of our results provides a
guideline for the design of pH-dependent protein-protein interactions. 相似文献
69.
70.